The G12/13 family of G proteins couple to tyrosine kinase and Rho signaling pathways and regulate numerous epithelial cell functions. Epithelial cell tight junctions (TJs) provide the paracellular barrier and maintain polarity. Disruption of TJs is an early (and reversible) event in ischemia/reperfusion (I/R) injury, and loss of TJs initiates additional pathways contributing to progressive interstitial fibrosis. We have made a number of observations regarding the role of G proteins in the regulation of tight junctions. We are now extending these observations into the role of G proteins and their regulation of tight junctions in acute renal injury. We identified direct binding of Ga12 with ZO-1, a TJ scaffolding protein, and elucidated Ga12-Src mediated signaling leading to TJ disruption. In addition, Ga12 and Ga13 interact with PP2A, a phosphatase within the TJ critical for regulating TJ assembly. We hypothesize that Ga12/13 regulate TJs through tyrosine kinase and Rho pathways to mediate early steps in the renal injury response. Therefore, Ga12 and Ga13 signaling are key therapeutic targets for regulating TJs and the recovery from acute kidney injury. In MDCK cells, activation of Ga12 and Ga13 leads to loss of barrier function and delayed TJ assembly through distinct mechanisms. Hypoxia-treated MDCK cells show delayed TJ assembly, and activated Ga13 is detected in kidney lysates from hypoxic mice. Our goals are to define Ga12/13 signaling mechanisms leading to loss of TJs, and how these pathways are modulated with renal injury. In Aim 1, the binding domains and the mechanisms regulating Ga12/ZO-1 and PP2A interactions will be completed using in vitro assays. In Aim 2, Ga12 and Ga13 signaling pathways through Src, PP2A and Rho regulating TJs will be studied in MDCK cells. Ga12 will be silenced to determine if loss of TJs can be prevented, and Ga12/13 signaling investigated in models of injury (ATP depletion and ROS). In Aim 3, animal models of hypoxia, I/R and ureteral obstruction will be used to determine Ga12/13 activation of Src and Rho pathways in renal tubules. A mouse with conditional expression of activated Ga12 in proximal tubules will be used to determine how Ga12 regulates TJs and renal tubular function. Kidney cells are tightly connected to prevent waste in the urine from reentering the body. These connections are injured in kidney disease, and these studies will determine how these connections are rebuilt after injury. This will aid discovery of new treatments to prevent kidney failure.